Lab 23: PCR and Gel Electrophoresis Flashcards
what is the purpose of PCR?
to amplifiy target DNA:
to create billions of copies of a specific region of DNA called the target DNA
what is PCR testing used for?
disease diagnosis
species identification
amplify DNA for cloning processes
what are the 3 steps of PCR?
- Denaturation
- Annealing
- Synthesis/Extension
what happens in denaturation?
-Specificity: to design primers complementary to the ends of target DNA sequence
-Denaturation: unzips the DS DNA by heating at 94 degrees Celsius for 1 minute
what happens in annealing?
Cooling down - which allows the primers to anneal or bind to the complementary sequences on target DNA - which is at 60 degrees Celsius for 1 minutes
what happens in synthesis/extension?
-The target DNA is copied by a thermostable DNA polymerase called TAQ - this is done at 72 degrees Celsius for 2 minutes.
-This uses the process of DNA replication
what do we get after steps 1-3? what do we do after?
We get 2 DS DNA molecules.
Then, we repeat steps 1-3 for 40 times
what is the result after this whole process including the 40 times repeated?
billions of target DNA copies
what are the components needed for a PCR reaction?
-Template DNA
-Primers for target DNA
-Taq DNA polymerase
-Nucleotides (dNTPs)
-Buffer
what is Taq DNA polymerase important for?
Because it is thermostable, it doesn’t denature at high temperatures from step 1
what is gel electrophoresis used for?
to detect PCR products
what is the gel that we pour?
We pour an agarose (polysaccharide) gel, which has a matrix-like structure with small pores through which DNA molecules can travel
what happens after we pour the gel?
we apply an electrical current - the DNA molecules run towards red (the positive electrode) as they are negatively charged
what size DNA molecules move faster through the gel?
smaller DNA molecules - separation based on size
what is the loading dye?
it is a dye that helps monitor the movement of samples; switch off electrical current when dye gets to the bottom
what is the molecular weight ladder?
a standard consisting of a mixture of DNA fragments with known sizes - it helps determine the size of an unknown DNA
what is SYBR green?
a dye that binds to the DNA allowing detection - it fluoresces when exposed to UV light
what was the goal of the lab?
to determine which one (A, B, or C) has Streptococcus pneumoniae 19A?
So we took the sputum samples from 3 patients - what did we do to the samples?
-grow cultures
-isolate DNA
-run PCR with both primer pairs
-compare with a (+) control of S. pneumoniae 19A to see which patient is infected with that strain
what two primers did we use?
Primer 1: to detect a gene present in all S. pneumoniae (all pt’s tested have S. pneumoniae gene).
Primer 2: to detect a gene present in only S. pneumoniae 19A strain (infected pt only has this S. pneumoniae 19A strain)
what does the control contain in the lab?
The control has BOTH primers, 1 and 2. So it has S. pneumoniae and S. pneumoniae 19A.
what are we trying to find out in the lab?
We are trying to see which pt has the S. pneumoniae 19A gene. AKA which pt has both bands from the C+.
slide 13
T/F: we can’t visualize the dye until it is under the UV
True
